1. Field of the Invention
There is a continuing and increasing need for accurate, sensitive techniques for measuring trace amounts of organic materials in a wide variety of samples. This need includes the measurement of drugs, naturally occurring physiologically active compounds or nutrients in physiological fluids, the presence of trace amounts of contaminants or toxic materials in foods, water or other fluids, and the like, as well as monitoring materials for trace contamination introduced during chemical processing.
Among the various techniques which have found increasing exploration are techniques involving receptors which recognize or bind to a specific polar and spatial organization of one or more molecules. For the most part, the receptors are antibodies and the techniques are referred to as immunoassays. These techniques conventionally employ a labeled ligand where the binding to the receptor allows for distinguishing between a bound labeled ligand and an unbound labeled ligand. Certain techniques, generally referred to as heterogeneous, rely on segregating the bound from the unbound labeled ligand. Other techniques, generally referred to as homogeneous, rely on the bound labeled ligand providing a signal level different from unbound labeled ligand.
In many of the homogeneous techniques, the label must interact with another substance in order to differentiate the signal. For example, in one technique, the label is an enzyme and when receptor is bound to the ligand the enzymatic activity is inhibited. This requires that the enzyme ligand combination be such that when receptor is bound to the enzyme ligand conjugate, either substrate is inhibited from entering the active site or the enzyme is allosterically modified, so that its turnover rate is substantially reduced. In another technique, a fluorescent label is employed in conjunction with a receptor for the fluorescer. The binding of the receptor to the fluorescer substantially diminishes the fluorescence when the fluorescer is irradiated with light which normally excites the fluorescer. When the fluorescer is conjugated to ligand, and antiligand is bound to the conjugate, the antifluorescer is inhibited from binding to the fluorescer.
While these techniques have found great use or show great promise, there is still an interest in enhancing the sensitivity of techniques which do not require separation. As lower and lower concentrations of analytes are encountered, improvements in available techniques are required to allow for accurate determination of the presence of extremely small amounts of the analyte. Therefore, there has been an ongoing effort to find new and improved ways to measure extremely small amounts of organic molecules in a wide variety of environments.
2. Description of the Prior Art
U.S. Pat. No. 3,817,837 describes a homogeneous enzyme immunoassay. U.S. Pat. No. 3,996,345 describes a homogeneous immunoassay employing two chromophores related by being a fluorescer and a quencher. Co-pending application Ser. No. 893,650, filed Apr. 5, 1978 describes a technique employing a plurality of enzymes, where the substrate of one enzyme is the product of the other enzyme. U.S. Pat. No. 3,935,074 describes an immunoassay involving steric hindrance between two antibodies. Co-pending application Ser. No. 815,487, describes an enzyme immunoassay, employing antienzyme as an inhibitor.